ES2247105T3 - METHODS TO PREPARE CLA ISOMERS. - Google Patents

Abstract

A method comprising: a) providing a first isomer of partially purified octadecadienoic acid having a conjugated binding system; and b) heating said first isomer of the partially purified octadecadienoic acid between 200 and 240 ° C so that said conjugate bonding system migrates, thereby forming a mixture comprising at least said first octadecadienoic acid isomer and a second octadecadienoic acid isomer, in that the first and second isomers of octadecadienoic acid are selected from the following list of pairs: first isomer second isomer c9, t11 t8, c10 t10, c12 c11, t13 c7, t9 t6, c8 t11, c13 c12, t14 c6, t8 t5, c6 c5, t7 t4, c6 c4, t6 t3, c5 t12, c14 c13, t15 t13, c5 c14, t16 t8, c10 c9, t11 c11, t13 t10, c12 t6, c8 c7, t9 c12, t14 t11, c13 t5, c6 c6, t8 t4, c6 c5, t7 t3, c5 c4, t6 c13, t15 t12, c14 c14, t16 t13, c5 and in which said second isomer is present in a concentration greater than 20% by weight of said first isomer.

Description

Methods for preparing CLA isomers.

Field of the Invention

The present invention relates to biochemistry of lipids, and in particular to the preparation of various isomers of conjugated linoleic acid.

Background of the invention

In 1978, researchers at the University of Wisconsin discovered the identity of a substance contained in cooked beef that seemed to inhibit mutagenesis. The substance was found to be a mixture of positional isomers of linoleic acid (C18: 2) with conjugated double bonds. The isomers c9, t11 and t10, c12 are present in greater abundance, but it is not true that the isomers are responsible for the biological activity observed. It has been observed in studies of marked absorption that the 9,11 isomer seems to be preferably somewhat related and incorporated into the phospholipid fraction of animal tissues and to a lesser extent in the 10,12 isomer (Ha, et al. , Cancer Res . , 50: 1097 [1990]).

The biological activity related to conjugated linoleic acids (called CLA) is diverse and complex. Currently, very little is known about the mechanisms of action, although several evolving preclinical and clinical studies are likely to shed new light on physiological and biochemical modes of action. The anticarcinogenic properties of CLA have been well documented. The administration of CLA inhibits the carcinogenesis of rat breasts, as demonstrated by Birt, et al. , Cancer Res. , 52: 2035s [1992]). Ha, et al. , supra , described similar results in a mouse neoplasm model of the mouse. CLA has also been identified as a potent cytotoxic agent against melanoma, human colorectal and breast cancers in vitro .

Although the mechanisms of action of CLA are still obscure, there is evidence that some component (s) of the immune system may be involved, at least in vivo . US Patent No. 5,585,400 (Cook, et al. , Incorporated herein by reference), discloses a method for attenuating allergic reactions in animals mediated by type I or IgE hypersensitivity by administering a diet containing CLA. It was also shown that CLA in concentrations of approximately 0.1 to 1.0 percent was an effective adjuvant for the conservation of white blood cells. US Patent No. 5,674,901 (Cook et al. , Incorporated herein by reference), disclosed that oral or parenteral administration of CLA in the form of free acid or salt resulted in elevation in lymphocyte subpopulations. CD-4 and CD-8 associated with cell-mediated immunity. The unfavorable effects that appear in pretreatment with the exogenous tumor necrosis factor could be indirectly alleviated by raising or maintaining the levels of CD-4 and CD-8 cells in animals to which CLA was administered. Finally, US Patent No. 5,430,066 (Cook, et al. , Incorporated herein by reference), describes the effect of CLA in preventing weight loss and immunostimulation anorexia.

Apart from the potential therapeutic and pharmacological applications of CLA indicated above, much enthusiasm has been raised regarding the use of CLA as a dietary supplement. It has been found that CLA exerts a profound generalized effect on body composition, in particular redirecting the distribution of fat and mass in lean tissue. US Patent No. 5,554,646 (Cook, et al. , Incorporated herein by reference), discloses a method that uses CLA as a dietary supplement by which pigs, mice and humans were fed diets containing 0 , 5 percent of CLA. In each species, a significant decrease in fat content was observed with a corresponding increase in protein mass. It is interesting that in these animals, the increase in the fatty acid content of the diet by the addition of CLA did not produce any increase in body weight, but was related to the redistribution of fat and lean inside the body. Another dietary phenomenon of interest is the effect of enrichment in CLA on the transformation of feed. US Patent No. 5,428,072 (Cook, et al. , Incorporated herein by reference), provided data demonstrating that the incorporation of CLA into animal feed (birds and mammals) increased the efficiency of feed transformation leading to greater weight gain in birds and mammals supplemented with CLA. The potential beneficial effects of CLA enrichment for animal growth feeds are evident.

Another important source of interest in CLA, and one that emphasizes its first commercial potential, is that which occurs naturally in food and feed consumed by humans and similar animals. In particular, CLA is abundant in products from ruminants. For example, several studies have been conducted in which CLA has been examined in various dairy products. Aneja, et al. , ( J. Dairy Sci ., 43: 231 [1990]) observed that the treatment of milk in yogurt produced a concentration of CLA. (Shanta, et al. , Food Chem. 47: 257 [1993]) demonstrated that a combined increase in treatment temperature and serum addition increased the concentration of CLA during the preparation of processed cheese. In an independent study, Shanta, et al. J. Food Sci. , (60, 695 [1995]) described that while treatment and storage conditions did not significantly reduce CLA concentrations, no increase was observed. In fact, several studies have indicated that seasonal or cross-animal variation can be taken into account in differences up to three times the CLA content of cow's milk (see eg, Parodi et al. , J. Dairy Sci . , 60: 1550 [1977]). Likewise, dietary factors have been implicated in the variation of the CLA content (Chin, et al. , J. Food Comp. Anal. 5: 185 [1992]). Due to this variation in the CLA content in natural sources, the ingestion of prescribed amounts of various foods will not guarantee that the individual or animal receives the optimal dose that ensures the achievement of the desired nutritional effect.

In the development of a defined commercial source of CLA for both therapeutic and nutritional applications, a procedure is needed for the generation of large quantities of the defined material. The problem with most CLA products prepared by conventional methods is their heterogenicity and substantial variation in the isoform from batch to batch. A recent publication documents this variation and indicates the need for CLA producers to assess the complex nature of their products (see Christie et al. , JAOCS , 74 (11): 1231 [1997]).

Considerable attention has been given to the fact that ingestion of large amounts of hydrogenated oils and solid fats, rather than animal tallow, results in high diets in trans fatty acid content. For example, Holman, et al. , ( Proc. Natl. Acad. Sci. , 88: 4830 [1991]) demonstrated that hydrogenated oils from rat feed resulted in an accumulation in the rat liver of unusual isomers of polyunsaturated fatty acids, which appear to interfere with metabolism of natural polyunsaturated fatty acids. Therefore, there is a strong need for chemical and biochemical analysis of the various isomers of CLA.

Compendium of the invention

The present invention relates to biochemistry of lipids, and in particular to the preparation of various isomers of conjugated linoleic (octadecadienoic acid). The present invention is not limited to the production of any particular isomer of octadecadienoic acid. In fact, you can prepare a variety of isomers that comprise, but are not limited to, c9, t11 acid octadecadienoic acid, t8, c10 octadecadienoic acid, t10, c12 octadecadienoic acid, c11, t13 octadecadienoic acid, c7 acid, t9 octadecadienoic acid, t6, c8 octadecadienoic acid, t11, c13 octadecadienoic acid, c12, t14 octadecadienoic acid, c6 acid, t8 octadecadienoic acid, t5, c6 octadecadienoic acid, c5, t7 octadecadienoic acid, t4, c6 octadecadienoic acid, t3, c5 octadecadienoic acid, t12, c14 octadecadienoic acid, acid octadecadienoic c13, t15 and c14 acid, t16 octadecadienoic. The Isomers can be supplied as free fatty acids, esters of alkyl or acyl glycerides. Therefore, in some embodiments, the present invention provides a method for the production of a composition comprising more than 25% of one of the above isomers. Still in other embodiments, the The present invention provides a method for the production of a composition comprising more than 40% of one of the isomers previous. In still other embodiments, the present invention provides a method for the production of a composition that It comprises more than 50% of one of the above isomers. Again, Isomers can be supplied as free fatty acids, alkyl esters or acyl glycerides. In some others preferred embodiments, the present invention provides a method for the production of a composition comprising more than 60% of CLA c11, t13.

In particularly preferred embodiments, the The present invention provides a method for the production of a mixture of acyl glycerols comprising residues of isomers of conjugated linoleic acid, in which the fatty acyl content of said acyl glycerols is at least 25% of an acid isomer conjugated linoleic selected from the group consisting of acid t8, c10 octadecadienoic acid, c11, t13 octadecadienoic acid, acid c7, t9 octadecadienoic acid, t6, c8 octadecadienoic acid, t11 acid, c13 octadecadienoic acid, c12, t14 octadecadienoic acid, c6 acid, t8 octadecadienoic acid, t5, c6 octadecadienoic acid, c5, t7 octadecadienoic acid, t4, c6 octadecadienoic acid, t3, c5 octadecadienoic acid, t12, c14 octadecadienoic acid, c13 acid, t15 octadecadienoic acid and c14, t16 octadecadienoic acid. In others particularly preferred embodiments, the present invention provides a method for the production of a mixture of acyl glycerols comprising acid isomer moieties conjugated linoleic, in which the fatty acyl content of said acyl glycerols is at least 45% of an isomer of conjugated linoleic acid selected from the group consisting of t8, c10 octadecadienoic acid, c11, t13 octadecadienoic acid, acid c7, t9 octadecadienoic acid, t6, c8 octadecadienoic acid, t11 acid, c13 octadecadienoic acid, c12, t14 octadecadienoic acid, c6 acid, t8 octadecadienoic acid, t5, c6 octadecadienoic acid, c5, t7 octadecadienoic acid, t4, c6 octadecadienoic acid, t3, c5 octadecadienoic acid, t12, c14 octadecadienoic acid, c13 acid, t15 octadecadienoic acid and c14, t16 octadecadienoic acid. Still in more particularly preferred embodiments, the present invention provides a method to produce a mixture of glycerols of acyl comprising residues of isomers of linoleic acid conjugate, in which the fatty acyl content of said acyl glycerols is at least 50% of an acid isomer conjugated linoleic selected from the group consisting of acid t8, c10 octadecadienoic acid, c11, t13 octadecadienoic acid, acid c7, t9 octadecadienoic acid, t6, c8 octadecadienoic acid, t11 acid, c13 octadecadienoic acid, c12, t14 octadecadienoic acid, c6 acid, t8 octadecadienoic acid, t5, c6 octadecadienoic acid, c5, t7 octadecadienoic acid, t4, c6 octadecadienoic acid, t3, c5 octadecadienoic acid, t12, c14 octadecadienoic acid, c13 acid, t15 octadecadienoic acid and c14, t16 octadecadienoic acid.

In other embodiments, the present invention provides a method of producing a composition comprising isomers of conjugated linoleic acid, in which the composition comprises or consists essentially of at least 25% of a first octadecadienoic acid isomer and at least 25% of an isomer brother of said first isomer. The present invention is not limited. to any pair of sister isomers. In fact, a variety of pairs of sister isomers that include, but are not limited to c9, t11 octadecadienoic acid and t8, c10 acid octadecadienoic acid, t10, c12 octadecadienoic acid and c11, t13 octadecadienoic acid, c7, t9 octadecadienoic acid and t6, c8 octadecadienoic acid, t11, c13 octadecadienoic acid and c12 acid, t14 octadecadienoic acid, c6, t8 octadecadienoic acid and t5, c6 octadecadienoic acid, c5, t7 octadecadienoic acid and t4, c6 octadecadienoic acid, c4, t6 octadecadienoic acid and t3, c5 octadecadienoic acid, t12, c14 octadecadienoic acid and c13, t15 octadecadienoic acid, and t13, c15 octadecadienoic acid and c14 acid, t16 octadecadienoic. Isomers can be provided as acids free fatty acids, alkyl esters or acyl glycerides.

In still other embodiments, the present invention provides methods for the preparation of pairs of octadecadienoic acid isomers and / or individual isomers of octadecadienoic acid comprising: a) providing a first partially purified octadecadienoic acid isomer; and b) treat the first isomer of octadecadienoic acid under conditions such that the conjugated link system migrates, forming from this mode a mixture containing at least the first and second isomers of octadecadienoic acid. Still in additional embodiments, the methods further comprise step c) separating at least the first and second isomers of octadecadienoic acid for provide a second isomer of octadecadienoic acid purified. The conditions include heating of the isomer of partially purified octadecadienoic acid. Still in other embodiments, the separation step is carried out by gas-liquid chromatography. In some embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids c9, t11 and t8, c10 octadecadienoic acid and the second isomer of octadecadienoic acid is the other of the acids t8, c10 and c9, t11 octadecadienoic acids. In some embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids t10, c12 and c11, t13 octadecadienoic acid and the second isomer of octadecadienoic acid is  the other of the acids t10, c12 and c11, t13 octadecadienoic acids. In some embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids c7, t9 and t6, c8 octadecadienoic acid and the second isomer of octadecadienoic acid is the other of the acids t10, c12 and c11, t13 octadecadienoic acids. In some embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids t11, c13 and c12, t14 octadecadienoic acid and the second isomer of octadecadienoic acid is another of the t11, c13 and c12, t14 octadecadienoic acids. In some embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids c6, t8 and t5, c6 octadecadienoic acid and the second isomer of octadecadienoic acid is the other of the acids c6, t8 and t5, c6 octadecadienoic acids. In others embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids c5, t7 and t4, c6 octadecadienoic acid and the second isomer of octadecadienoic acid is the other of the acids c5, t7 and t4, c6 octadecadienoic acids. In some embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids t3, c5 and c4, t6 octadecadienoic acid and the second isomer of octadecadienoic acid is the other of the acids c4, t6 and t3, c5 octadecadienoic acids. In some embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids t12, c14 and c13, t15 octadecadienoic acid and the second isomer of octadecadienoic acid is another of the acids t12, c14 and c13, t15 octadecadienoic acids. In others embodiments, the first isomer of octadecadienoic acid partially purified is one of the acids c14, t16 and t13, c15 octadecadienoic acid and the second isomer of octadecadienoic acid is the other of said acids c14, t16 and t13, c15 octadecadienoic acids.

In some embodiments, the second isomer is present in a concentration greater than 30% of the first isomer. In other embodiments, the second isomer is present in a concentration greater than 40% of the first isomer. Still in others embodiments, the second isomer is present in a concentration between 20% and 80% of the first isomer.

As used herein, "acid conjugated linoleic "or" CLA "refers to any acid conjugated linoleic or free octadecadienoic fatty acid. Be It is intended that this expression encompasses and indicates all isomers of position and geometric linoleic acid with two double bonds carbon-carbon conjugates anywhere in the molecule. CLA differs from ordinary linoleic acid in that the Ordinary linoleic acid has double bonds in the atoms of carbon 9 and 12. Examples of CLA include cis and trans isomers. ("E / Z isomers") of the following position isomers: acid 2,4-octadecadienoic acid 4,6-octadecadienoic acid 6,8-octadecadienoic acid 7,9-octadecadienoic acid 8,10-octadecadienoic acid 9,11-octadecadienoic acid 10,12-octadecadienoic acid 11,13-octadecadienoic. As used in this memory, "CLA" comprises a single isomer, a mixture selected from two or more isomers and an unselected mixture of isomers obtained from natural sources, as well as synthetic CLA and semi-synthetic.

As used herein, the expression "isomerized conjugated linoleic acid" refers to CLA synthesized by chemical methods (e.g., alkali isomerization aqueous, isomerization in non-alkali alkali or isomerization in alkaline alcoholate).

As used herein, the expression "conjugated linoleic acid group" refers to any compound or plurality of compounds containing acids or conjugated linoleic derivatives. The examples include, but not limited to fatty acids, alkyl esters and triglycerides of conjugated linoleic acid.

As used herein, it is intended that the "triglycerides" of CLA contain CLA in any or the three positions (e.g., SN-1 positions, SN-2 or SN-3) on the central axis of the glycerol. Therefore, a triglyceride containing CLA can contain any of the positional and geometric isomers of CLA. As used herein, "acylglycerol" is refers to a glycerol molecule that has a (monoacrylic glycerol), two (diacylglycerol) or three (triacylglycerol or triglyceride) residues fatty acyl in any of the positions available in The glycerol molecule. Triglyceride mixtures or acylglycerols may contain varying amounts of certain CLA isomers depending on the concentration of certain isomers in the CLA used to produce triglyceride and, if use enzymatic procedures to prepare triglyceride or Acylglycerol, the specificity of the lipase or lipases used. Therefore, it is convenient to characterize the mixture or resulting triglyceride or acylglycerol composition depending on the amount, referred in percentage, of a given isomer of CLA contained within the mixture or complete composition. In title non-limiting example, CLA residues containing the composition of triglyceride or acylglycerol in various positions in the central axis of glycerol can be said to have a content rest of fatty acyl of at least 25% of a given conjugated linoleic acid isomer when this acid isomer Conjugated linoleic occupies at least 25% of any of the positions SN-1, SN-2 and SN-3 of the triglycerides in the mixture. Seen from otherwise, if the fatty acyl residues are removed by saponification, the resulting mixture of fatty acid salts It will comprise at least 25% of the determined isomer.

As used herein, it is intended that the "esters" of CLA include any and all isomers CLA position and geometries joined by an ester link to an alcohol or any other chemical group, including, but not limited to natural, physiologically acceptable alcohols (e.g., methanol, ethanol or propanol). Therefore, an ester of CLA or an esterified CLA may contain any of the isomers CLA position and geometric.

It is intended that "unnatural isomers" of CLA include, but are not limited to c11, t13 isomers; t11, c13; t11, t13; c11, c13; c8, t10; t8, c10; t8, t10; c8, c10; Y trans-trans octadecadienoic acid. But nevertheless, This definition does not include the t10, c12 and c9, t11 isomers of the acid octadecadienoic. "Unnatural isomers" may also be called "minor isomers" of CLA, since these isomers are generally produced in small quantities when synthesized CLA by isomerization with alkali.

As used herein, CLA of "low impurity" refers to CLA compositions, which include free fatty acids, alkyl esters and triglycerides, containing less than 1% total 8,10 octadecadienoic acids, 11,13 octadecadienoic acids or trans-trans acids octadecadienoic.

As used herein, "c" it comprises a chemical bond in the cis orientation, and "t" is refers to a chemical bond in the trans position. If an isomer of CLA position is designed without a "c" or a "t", then This designation includes the four possible isomers. By example, 10.12 octadecadienoic acid comprises c10, t12 acid; t10, c12; t10, t12 and c10, c12 octadecadienoic acid while the acid t10, c12 octadecadienoic or CLA refers only to a single isomer

As used herein, the term "oil" refers to a freely flowing liquid that Contains long chain fatty acids (e.g. CLA), triglycerides or other long chain hydrocarbon groups. Fatty acids from Long chain include, but are not limited to the various isomers of CLA.

As used herein, the expression "physiologically acceptable carrier" refers to any carrier or excipient normally used with products oily pharmacists Such carriers or excipients include, but are not limited to, oils, starches and sugars (e.g., sucrose and lactose).

As used herein, the expression "oral administration vehicle" refers to any means to administer a pharmaceutical product orally, including but not limited to capsules, pills, tablets and syrups.

As used herein, the expression `` food product refers to any food or feed suitable for consumption by humans, non-ruminant animals or ruminant animals The "food product" can be a prepared and packaged food (e.g., mayonnaise, dressing salad, bread or cheese) or animal feed (e.g. animal feed extruded and in granules or of course mixed feed). "Product prepared food "means any prepackaged food approved for human or animal consumption.

As used herein, the expression "edible product" refers to any substance suitable for human or animal consumption.

As used herein, the expression "volatile organic compound" refers to any compound containing carbon that exists partially or completely in a state gas at a given temperature. Volatile organic compounds they can be formed in the oxidation of an organic compound (e.g., CLA). Volatile organic compounds include, but are not limited to pentane, hexane, heptane, 2-butenol, ethanol, 3-methyl butanal, 4-methyl pentanone, hexanal, heptanal, 2-pentyl furan and octanal

As used herein, the expression "metal chelating oxidant" refers to any antioxidant that sequesters metals. Examples include, but not they are limited to lecithins and citric acid esters.

As used herein, the expression "partially purify" means any process that remove some of a pollutant from the component of interest, such as unwanted removal of isomers from an acid preparation conjugated linoleic. The percentage of a purified component thus increases in the sample (i.e., the component of interest).

As used herein, the term "sister isomers" refers to pairs of conjugated isomers of linoleic acid that can be interconverted by some treatment (eg, heating). Examples of sister isomers are given in Table 1, infra .

As used herein, the expression "conjugate link system" refers to the following arrangement of unsaturated double bonds in a chain Carbohydrate: -C = C-C = C- (that is, two doubles links separated by a simple link).

As used herein, the term "migrate", when used in reference to a system of conjugated link in carbohydrate chains, refers to the disposition of carbon-carbon double bonds saturated within the carbohydrate chain so that the double bonds move up and down the chain.

Detailed description of the invention

The present invention relates to the biochemistry of lipids, and in particular to the preparation of various isomers of conjugated linoleic acid. It has been shown that reconfiguration of the double bonds of linoleic acid to conjugated positions takes place during treatment with catalysts such as nickel or alkali at high temperatures, and during autooxidation. Theoretically, eight possible geometric isomers of 9,11 and 10,12 octadecadienoic acid (c9, c11; c9, t11; t9, c11; t9, t11; c10, c12; c10, t12; t10, c12 and t10, t12) they would form from the isomerization of c9, c12-octadecadienoic acid. A general mechanism for isomerization of linoleic acid was described by JC Cowan ( JAOCS 72: 492-99 [1950]). It is believed that the double bond is polarized as a result of a collision with an activation catalyst. The polarized carbon atom and its adjacent carbon are then free to rotate and the forces are such as to make the deficient carbon atom essentially flat. When the system then moves to release these forces created as a result of the collision, both cis and trans isomers are formed. The formation of certain CLA isomers is thermodynamically favored. This is due to the coplanar characteristics of the five carbon atoms around the conjugated double bond and a spatial conflict of the radical resonance. The relatively greater distribution of the 9,11 and 10,12 isomers apparently results from the further stabilization of the geometric isomers c9, t11 or t10, c12.

Advances in gas chromatography have allowed researchers to accurately determine the composition of the isomer of CLA samples. These studies show that many more than 8 isomers are actually formed during conjugation. In Christie et al. , ( JAOCS 74 (11): 1231 [1997]), it was described that the distribution of the isomer of a commercial CLA sample was as follows: 8.10 (14%); 9.11 (30%); 10.12 (31%) and 11.13 (24%). In another study by Christie et al. in ( Lipids 33 (2): 217-21 [1998]), the composition of the following CLA isomer of a commercial CLA preparation was described: t11, t13 (0.74%); t10, t12 (1.23%); t9, t11 (1.18%); t8, t10 (0.46%); c11, t13 and t11, c13 (21.7%) c10, t12 and t10, c12 (29.0%); c9, t11 and t9, c11 (29.5%); c8, t10 and t8, c10 (12.3%); c11, c13 (0.96%); c10, c12 (0.88%); c9, c11 (0.88%); and c8, c10 (0.20%). As can be seen in these studies, even when the formation of certain isomers is favored, other CLA isomers can contribute greatly to the composition of CLA preparations isomerized in alkali.

A. Migration of conjugated double bonds

The present invention provides methods for produce enriched compositions for different isomers of CLA. In some embodiments of the invention, a CLA isomer partially purified or concentrated is treated under conditions that Double link system migration occurs. In the preferred embodiments, the conditions comprise the heating of at least one isomer to approximately 200-240 ° C, preferably at about 220 ° C. In other embodiments, the conditions further comprise making react the isomer or partially purified isomers or nitrogen concentrates in a sealed container. With reference to Table 1, preparations of the isomers can be used of column 1 to produce preparations containing a substantial amount of the corresponding isomer of column 2. After the initial conversion reaction, the preparation will contain both the starting isomer and the "brother" isomer. Also, preparations of the isomers of column 2 are they can use to produce substantial amounts of corresponding isomer of column 1. The preparations that contain both isomers can also be treated to purify the sister isomer (e.g., by gas chromatography). How Experts in the field will understand, it is possible to start from more than a partially purified isomer, thereby producing a preparation containing four, six, eight or more isomers. In other embodiments, a purified preparation can be prepared of the sister isomer by methods known in the art (i.e. gas-liquid chromatography) of the treated preparation containing the initial isomer and its sister isomer.

TABLE 1

Column one Column 2 c9, t11 t8, c10 t10, c12 c11, t13 c7, t9 t6, c8 t11, c13 c12, t14

TABLE 1 (continued)

Column one Column 2 c6, t8 t5, c6 c5, t7 t4, c6 c4, t6 t3, c5 t12, c14 c13, t15 t13, c15 c14, t16

As demonstrated in the Examples, the treatment of t10, c12 octadecadienoic acid resulted in production of c11, t13 octadecadienoic acid. It can also be used c11, t13 octadecadienoic acid concentrated or partially purified to produce t10, c12 octadecadienoic acid. After of treating the partially purified isomer to produce a sister isomer, the resulting preparation comprises with preference approximately more than 20% of the sister isomer in comparison with the partially purified initial isomer. In others   embodiments, the resulting preparation preferably comprises approximately more than 40% of the sister isomer compared to the initial isomer partially purified. Still in others embodiments, the resulting preparation comprises approximately between approximately 20% and 80% of the sister isomer in comparison with the partially purified initial isomer. Still In other embodiments, the resulting preparation comprises or It consists essentially of more than about 20% of the isomer brother in weight / weight (e.g., the preparation contains approximately 20 grams of the sister isomer and approximately 80 grams of the partially purified initial isomer). In others embodiments, the resulting preparation comprises approximately more than 40% of the sister isomer by weight / weight. Still in others embodiments, the preparation comprises between about 20% and 80% of the sister isomer by weight / weight.

Experimental part

The following examples are provided in order to demonstrate and illustrate more certain preferred embodiments and aspects of the present invention and should not be considered limiting its scope.

In the experimental exhibition that follows, it the following abbreviations apply: M (molar); mM (millimolar); µM (micromolar); kg (kilograms); g (grams); mg (milligrams); \ mug (micrograms); ng (nanograms); l (liters); ml (milliliters); mul (microliters); cm (centimeters); mm (millimeters); nm (nanometers); ºC (degrees Celsius); KOH (potassium hydroxide); HCl (hydrochloric acid); and Hg (mercury).

Example 1 Preparation of CLA isomers

This Example describes acid production c11, t13 octadecadienoic acid from t10, c12 octadecadienoic. Fifty grams of KOH was dissolved in Propylene glycol with moderate heating. They were added to then to the mixture one hundred grams of 98% linoleic acid, it the mixture was heated to 150 ° C and stirred for 3 hours. It cooled to then the mixture and washed several times with hot water and to It was then dried under vacuum with moderate heat. CLA mix resulting consisted of c9, t11 and t10, c12 octadecadienoic acid as well as from vestiges of CLA isomers. The mixture became boiling methyl ester at reflux in acid methanol. Be dissolved fifty grams of conjugated free fatty acids in methanol containing 4.5% sulfuric acid and boiled in reflux conditions for 1 hour in a water bath. He cooled the mixture and the bottom layer was discarded. Methanol was added fresh with 4.5% sulfuric acid in the boiled mixture for another hour under reflux conditions. After cooling, this methyl ester mixture was washed several times with water and to It was then dried under vacuum with moderate heat. They dissolved ten grams of the methyl ester in acetone and cooled during overnight at -60 ° C in a freezer. A precipitate was recovered solid by filtration and dissolved again in acetone and again cooled to -30 ° C overnight. The precipitate was dried at vacuum and was demonstrated by GLC analysis containing 97% of t10, c12 CLA. The analytical team consisted of a Perkin Elmer GLC with automatic sampler The column was of the molten silica type very polar The following equipment was used for the program:

Injection:

No separation at 250 ° C

Detection:

Flame ionization detector at 280ºC

Carrier:

Helium to psig.

Program of the stove:

80ºC-130ºC (45ºC / min), then 1ºC / min at 220ºC and ºC until the end for 10 min.

Column:

SILOT WCOT FOUND 0.25 mm x 100 m, CP-SIL 88 for FAME, df = 0.2.

One gram of isomer was covered with nitrogen t10, c12 purified then in a sealed tube and heated for two hours at 220 ° C. After cooling, the resulting methyl esters by GC as above. He relative content of t10, c12 in the mixture was reduced to 52.32% and the c11, t13 isomer was present in a concentration of 41.96% (see Table 2).

TABLE 2

Conversion of isomer t10, c12 in the isomer c1, t13 Isomer Before heating,% Behind the heating,% c11, t13 0 41.57 t10, c12 97.34 51.72 c11, c13 0 1.44 c10, c12 0 2.70 t11, t13 0 0.54 t10, t12 0.7 1.05

Example 2 Preparation of CLA isomers

This Example describes acid production t8, c10 octadecadienoic acid from c9, t11 octadecadienoic acid. Purified c9, t11 octadecadienoic acid can be purchased in commercial suppliers (Matreya, State Collage, PA) or through fermentation of ruminant microorganisms (see, e.g., the U.S. patent No. 5,674,901, incorporated herein as reference). The purified c9, t11 octadecadienoic acid is converts a large percentage (e.g., 25% to 50%) into acid t8, c10 octadecadienoic acid placing c9, t11 octadecadienoic acid in a tube sealed under nitrogen and heating at 220 ° C for about 2 hours

Example 3 Preparation of CLA isomers

This Example describes acid production t6, c8 octadecadienoic acid from c7, t9 octadecadienoic acid. He C7, t9 octadecadienoic acid purified can be obtained by gas chromatography at preparation scale. C7 acid, t9 purified octadecadienoic acid is transformed into a large percentage (e.g. eg, 25% to 50%) in t6, c8 octadecadienoic acid by placing the acid c9, t11 octadecadienoic in a tube sealed under nitrogen and heating at 220 ° C for approximately 2 hours.

Example 4 Preparation of CLA isomers

This Example describes acid production c12, t14 octadecadienoic acid from t11, c13 octadecadienoic. The purified t11, c13 octadecadienoic acid is can be obtained by gas chromatography on a preparation scale (e.g. eg, following the procedure described in Example 1). Acid t11, c13 purified octadecadienoic is transformed into a large percentage (e.g., 25% to 50%) in c12, t14 octadecadienoic acid placing c9, t11 octadecadienoic acid in a sealed tube under nitrogen and heating at 220 ° C for approximately 2 hours.

Example 5 Preparation of CLA isomers

This Example describes acid production t5, c6 octadecadienoic acid from c6, t8 octadecadienoic acid. He C6, t8 octadecadienoic acid purified can be obtained by gas chromatography at preparation scale. C6 acid, t8 purified octadecadienoic acid is transformed into a large percentage (e.g. e.g., 25% to 50%) in t5, c6 octadecadienoic acid by placing the acid c9, t11 octadecadienoic in a tube sealed under nitrogen and heating at 220 ° C for approximately 2 hours.

Example 6 Preparation of CLA isomers

This Example describes acid production t4, c6 octadecadienoic acid from c5, t7 octadecadienoic acid. He C5, t5 octadecadienoic acid purified can be obtained by gas chromatography at preparation scale. Acid C5, T7 purified octadecadienoic acid is transformed into a large percentage (e.g. e.g., 25% to 50%) in t4, c6 octadecadienoic acid by placing the acid c9, t11 octadecadienoic in a tube sealed under nitrogen and heating at 220 ° C for approximately 2 hours.

Example 7 Preparation of CLA isomers

This Example describes acid production t3, c5 octadecadienoic acid from c4, t6 octadecadienoic acid. He C4, t6 octadecadienoic acid purified can be obtained by gas chromatography at preparation scale. C4, T6 acid purified octadecadienoic acid is transformed into a large percentage (e.g. 25% to 50%) in t3, c5 octadecadienoic acid by placing the acid c9, t11 octadecadienoic in a tube sealed under nitrogen and heating at 220 ° C for approximately 2 hours.

Example 8 Preparation of CLA isomers

This Example describes acid production c13, t15 octadecadienoic acid from t12, c14 octadecadienoic. The purified t12, c14 octadecadienoic acid is can be obtained by gas chromatography on a preparation scale. He purified t12, c14 octadecadienoic acid is transformed into a large percentage (e.g., 25% to 50%) in c13, t15 octadecadienoic acid placing c9, t11 octadecadienoic acid in a sealed tube under nitrogen and heating at 220 ° C for approximately 2 hours.

Example 9 Preparation of CLA isomers

This Example describes acid production c14, t16 octadecadienoic acid from t13, c15 octadecadienoic. The purified t13, c15 octadecadienoic acid is can be obtained by gas chromatography on a preparation scale. He purified t13, c15 octadecadienoic acid is transformed into a large percentage (e.g., 25% to 50%) in c14, t16 octadecadienoic acid placing c9, t11 octadecadienoic acid in a sealed tube under nitrogen and heating at 220 ° C for approximately 2 hours.

Example 10 Preparation of CLA triacylglycerols by esterification direct

Immobilized antarctic Candida lipase (1.25 g) is added to a mixture of glycerol (1.22 g, 13.3 mmol) and desired CLA isomer. The mixture is stirred gently on a heating plate with magnetic stirrer at 65 ° C in a continuous vacuum of 0.01 to 0.5 Torr. The volatile water produced during the evolution of the reaction is continuously condensed in traps cooled with liquid nitrogen. After 48 h the reaction is interrupted, n-hexane is added and the enzyme is filtered off. The organic phase is treated with an aqueous alkaline solution of sodium carbonate to remove excess free fatty acids (when necessary). The organic layer is removed in vacuo (after drying over anhydrous magnesium sulfate where appropriate) in a rotary evaporator followed by high vacuum treatment to produce the practically pure product. When stoichiometric amounts of free fatty acids are used, standardized sodium hydroxide titration is applied to determine the free fatty acid content of the crude reaction product.

Example 12 Triacylglyceride production

CLA isomers are prepared as described. above and distilled in a molecular distillation plant at 150 ° C and at a pressure of 10-2 mbar. Then they mix 1,000 kg of the distilled product with 97 kg of pure glycerol and 80 kg of lipase The reaction is allowed to continue for 12 hours at 55 ° C at empty and in agitation. The triacylglyceride product is distilled in a molecular distillation apparatus to remove fatty acids without reacting

Example 13 Synthesis of c11, t13 CLA

This Example describes the production of a CLA sample containing approximately 60% of the c11, t13 isomer of CLA. In summary, two portions of a mixture of t10 fatty acid ethyl ester, 95% c12, 30 g each, a 70 ml test tubes. The tubes were purged with nitrogen rehearsal and sealed. The test tubes were preheated during one minute in a 100 ° C water bath and then placed directly in an oil bath at 220 ° C. After two hours, it He removed one of the test tubes from the oil bath and left cool. The other test tube was removed after another hour and then allowed to cool. GC were determined by Isomer profiles of the initial sample and two samples hot (Table 13).

Three samples were taken from each of the two hot samples and diluted in 3.5, 4.5 or 5.5 ml of Acetone per gram of fat in 70 ml test tubes (Table 14). The tubes were then cooled to -60 ° C in a freezer test with acetone solution. After 3 days, they were removed samples of the supernatant fractions. It was determined by GC the Isomer profile of the supernatant fractions (Table 15).

The heating of the initial sample (approximately 99% total CLA, 0.9% oleic acid) at 220 ° C for 2 and 3 hours it produced a partial conversion of the isomer t10, c12 in the isomer c11, t13. Hot samples contained 35 to 45% of isomer c11, t13 (Table 13). The exact content of the isomer could not be determined due to incomplete separation in GC conditions selected.

The crystallization of the heated sample during two hours differed from the crystallization of the heated sample for three hours by providing better (more solid) crystals. The sample heated for 3 hours provided medium crystals melted The analysis of the supernatant fractions put manifested that the crystallization of the heated sample during two hours gave a better separation with a higher content of c11,13 in the supernatant fraction. The content of CLA c11, t13 and t10, c12 in the supernatant fractions it was from 56 to 60% and from 27 to 30% respectively. The content of c11, t13 was slightly higher in the supernatant fractions of the samples with the proportion lower fat / acetone (Table 15).

TABLE 13

Fatty acid composition of the samples initials and hot * Sample without GC c9, t11 c11, t13 t10, c12 c10, c12 c11, c13 t11, t13 others t t Initial 5723 2.8 nd 94.8 0.8 0.2 nd 0.7 220 ° C 5724 2.8 38 55 0.7 1.4 0.3 0.8 2 h 220 ° C 5725 2.6 46 46 1.1 1.9 0.4 1.0 3 h *  \ begin {minipage} [t] {155mm} In hot samples, the values given in column c9, t11 represent c9, t11 and t8, c10 due to a similar conversion from c9, t11. The values given for the quantities of c10, c12 and c11, c13 are the values integrated by the GC. These values can be diverted from the content. actual due to incomplete separation in the chromatograms. \ end {minipage}

TABLE 14

Dilution of the initial sample in acetone before cooling* Dilution nº Amount of fat (g) Amount of Acetone (ml) one (1: 3.5) 7.0 24.5 2 (1: 4,5) 5.5 24.8 3 (1: 5.5) 4,5 24.8 * The two hot samples are diluted in equal amounts.

TABLE 15

Fatty acid profile of the fractions supernatants * Sample without GC c9, t11 c11, t13 t10, c12 c10, c12 c11, c13 t11, t13 others t t 2 h 5726 4.3 56 31 1.0 2.4 0.7 1.1 dil. one 2 h 5727 4.8 60 28 1.2 2.4 0.6 1.1 dil. 2 2 h 5728 5.6 59 27 1.2 3.0 0.6 1.1 dil. 3 3 h 5729 2.8 41 fifty 1.2 2.0 0.5 1.1 dil. one 3 h 5733 3.2 48 42 1.2 2.3 0.7 1.3 dil. 2 3 h 5734 3.7 56 33 1.3 2.6 0.6 1.1 dil. 3 *  \ begin {minipage} [t] {155mm} In hot samples, the values given in column c9, t11 represent c9, t11 and t8, c10 due to a similar conversion from c9, t11. The values given for the quantities of c10, c12 and c11, c13 are the values integrated by the GC. These values can be diverted from the content. actual due to incomplete separation in the chromatograms. \ end {minipage}

From the above it is evident that the The present invention provides methods for the preparation of CLA isomer compositions.

Claims (6)

1. A method comprising:

to)

provide a first isomer of partially purified octadecadienoic acid presenting a conjugated link system; Y

b)

heating said first acid isomer partially purified octadecadienoic between 200 and 240 ° C so that said conjugated link system migrates, thus forming a mixture comprising at least said first acid isomer octadecadienoic acid and a second isomer of octadecadienoic acid, in that the first and second isomers of octadecadienoic acid are Select from the following list of pairs:

first isomer second isomer c9, t11 t8, c10 t10, c12 c11, t13 c7, t9 t6, c8 t11, c13 c12, t14 c6, t8 t5, c6 c5, t7 t4, c6 c4, t6 t3, c5 t12, c14 c13, t15 t13, c5 c14, t16 t8, c10 c9, t11 c11, t13 t10, c12 t6, c8 c7, t9 c12, t14 t11, c13 t5, c6 c6, t8 t4, c6 c5, t7 t3, c5 c4, t6 c13, t15 t12, c14 c14, t16 t13, c5

and in which said second isomer is present in a concentration greater than 20% by weight of said first isomer.

2. The method according to claim 1, which it also includes the stage of

C)

separation of said first isomer from partially purified octadecadienoic acid and said second octadecadienoic acid isomer to provide a second partially purified octadecadienoic acid isomer.

3. The method according to claim 2, wherein said purification step is carried out by chromatography of gases or liquids. 4. The method according to claim 1, wherein said isomer of octadecadienoic acid comprises an ester of I rent. 5. The method according to claim 1, which it further comprises the synthesis of an acylglycerol with said mixture that it comprises at least said first octadecadienoic acid isomer and a second isomer of octadecadienoic acid. 6. The method according to claim 5, wherein said acylglycerol is a triglyceride.